Table 1.

Performance characteristics of a multiplexed LC-MRM/MS assay.

ISpepaISprotb
rcImprecision, % CVrImprecision, % CV
LinearitydLC-MSeDigestfTotalgLinearityLC-MSDigestTotal
A-I0.790.99822.69.49.80.960.99945.62.36.1
B0.670.99836.42.66.90.610.99758.93.59.6
C-II0.900.99484.14.66.10.920.99376.14.17.4
C-III0.890.99629.55.010.70.880.999211.84.812.8
E0.960.99842.23.34.00.920.99842.22.13.1
J0.810.99949.36.811.50.790.999112.43.312.8
  • a Isotope-labeled peptides were spiked after digestion and used as internal standards in a multiplexed LC-MRM/MS assay to quantify the relative concentration of 6 proteins in HDL (normalized MRM–peptide ratio).

  • b 15N isotope-labeled apoA-I was spiked prior to digestion and used as an internal standard in the LC-MRM/MS assay for the same 6 proteins (normalized MRM–protein ratio).

  • c Pearson correlation coefficient (r) of the relative protein concentration determined by LC-MRM/MS compared with immunoassay (n = 30).

  • d Linearity determined across a 4-point dilution series of human HDL into mouse HDL.

  • e Imprecision of the LC-MS step was determined by injecting digested pooled HDL 35 times over 3 days and 4 days later 24 times over 2 days. The mean imprecision of the 2 experiments is presented.

  • f Five separate digestions of HDL were injected 4 times (twice on each of days 1 and 9). The mean of the 4 injections was used as the relative concentration. The imprecision of the 5 digestions is presented.

  • g Total imprecision was estimated from the variability of the digestion and LC-MS steps (see online Supplemental Material).